Abstract
The molecular roles of many RNA-binding proteins in bacterial post-transcriptional gene regulation are not well understood. Approaches combining in vivo UV crosslinking with RNA deep sequencing (CLIP-seq) have begun to revolutionize the transcriptome-wide mapping of eukaryotic RNA-binding protein target sites. We have applied CLIP-seq to chart the target landscape of two major bacterial post-transcriptional regulators, Hfq and CsrA, in the model pathogen Salmonella Typhimurium. By detecting binding sites at single-nucleotide resolution, we identify RNA preferences and structural constraints of Hfq and CsrA during their interactions with hundreds of cellular transcripts. This reveals 3'-located Rho-independent terminators as a universal motif involved in Hfq-RNA interactions. Additionally, Hfq preferentially binds 5' to sRNA-target sites in mRNAs, and 3' to seed sequences in sRNAs, reflecting a simple logic in how Hfq facilitates sRNA-mRNA interactions. Importantly, global knowledge of Hfq sites significantly improves sRNA-target predictions. CsrA binds AUGGA sequences in apical loops and targets many Salmonella virulence mRNAs. Overall, our generic CLIP-seq approach will bring new insights into post-transcriptional gene regulation by RNA-binding proteins in diverse bacterial species.